KR20210067776A - Mass Manufacturing Method for Natural Killer Cells comprising Freezing and Thawing Process - Google Patents

Abstract

The present invention relates to a mass production method of natural killer cells. The mass production method of the present invention can produce natural killer cells in sufficient number for clinical application even through a cryopreservation process is undergone, thereby being usefully used for enhancing the prevention and treatment effects of cancer using natural killer cells, in particular, for enhancing the effect of allopathic treatment.

Description

Mass Manufacturing Method for Natural Killer Cells comprising Freezing and Thawing Process

The present invention relates to a method for mass production of natural killer cells including freezing and thawing.

The human body is protected from pathogens by an immune response, and the immune system is composed of many immune-related cells and cytokines. White blood cells, especially lymphocytes, play an important role in this immune system. Cells constituting lymphocytes are typically the innate immune system and the acquired immune system.

Natural Killer Cell (NK cell) is one of the representative innate immune cells that can kill cancer non-specifically, recognize viruses and bacteria, and kill them, including perforin and granzyme. It is known as a cell that kills pathogens through enzyme or Fas-FasL interaction. For cancer patients, the reduction in the cancer cell killing ability of these natural killer cells (NK cells) is lung cancer (Carrega P, et al., Cancer, 2008: 112: 863-875), liver cancer (Jinushi M, et al., J Hepatol., 2005: 43; 1013-1020), breast cancer (Bauernhofer T, et al., Eur J Immunol., 2003: 33: 119-124), cervical cancer (Mocchegiani E., et al., Br j Cancer., 1999: 79: 244-250), blood cancer (Tajima F., et al, Lekemia 1996: 10: 478-482) is reported to be closely related to the occurrence of diseases, and for the treatment of cancer, cancer patients It would be essential to increase the cancer cell killing ability and activity of natural killer cells.

In order to obtain this cancer-killing effect, a large amount of high-purity natural killer cells are required, but it is not easy to obtain a large amount of blood from cancer patients, and the proportion of natural killer cells in the blood is only about 5-20%. Therefore, it is difficult to use it as an immunotherapeutic agent.

Therefore, it is important to effectively expand and proliferate only natural killer cells, but the existing natural killer cell proliferation method requires the use of various expensive cytokines in high concentrations, so there is a problem that only some economically stable patients can be treated. In addition, other types of immune cells than natural killer cells (e.g., T cells, B cells, etc.) exist together, and when administered to allogenes containing T cells, it can cause GVHD (graft versus host disease), and B cells There is a problem in that the anticancer effect cannot be maximized, such as may cause transient lymphocyte syndrome (Passenger B-lymphocyte syndrome) when administered to allogenes of blood type incompatibility.

Domestic Patent Publication No. 10-2013-0086820

The present inventors made intensive research efforts to develop a method for mass production of natural killer cells through the cryopreservation process, in consideration of the production manpower, facilities, and equipment, etc. comprehensively. As a result, when CD56+ cells were co-cultured with irradiated feeder cells in the presence of cytokines, the present invention was completed by identifying that cell culture was smoothly performed even after freezing and thawing.

Accordingly, an object of the present invention is to provide a method for mass production of natural killer cells.

The present inventors made intensive research efforts to develop a method for mass production of natural killer cells through the cryopreservation process, in consideration of the production manpower, facilities, and equipment, etc. comprehensively. As a result, it was confirmed that, when CD56+ cells were co-cultured with irradiated feeder cells in the presence of cytokines, cell culture was performed smoothly even after freezing and thawing.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention relates to a method for mass production of natural killer cells comprising the steps of:

A first culture step of co-culturing at least one selected from the group consisting of CD56+ cells and CD3-/CD56+ cells with feeder cells and a first cytokine;

freezing;

thawing; and

A second culturing step of co-culturing by adding feeder cells.

Each step of the present invention will be described in detail.

first incubation step

As used herein, the term "CD56+ cell" may be used interchangeably with "CD56+ NK cell" or "CD56 natural killer cell", and "CD3-/CD56+ cell" may be used interchangeably with "CD3-/CD56+ NK cell". can

The CD56+ cells and CD3-/CD56+ cells may include, if necessary, cells expressing cell surface CD56 glycoprotein, and cells expressing CD56 glycoprotein but not expressing CD3 glycoprotein. Even the same immune cells may have different functions due to differences in the type and expression rate of CDs attached to the cell surface.

The CD56+ cells and CD3-/CD56+ cells can be obtained through the following steps.

A first separation step of isolating peripheral blood mononuclear cells (PBMC) from the blood sample; and

A second separation step of isolating any one or more selected from the group consisting of CD56+ cells and CD3-/CD56+ cells.

As used herein, "blood sample" may be peripheral blood, whole blood, or leukocytes isolated from peripheral blood using Leukapheresis, but is not limited thereto. In addition, the peripheral blood may be obtained from a normal person, a patient having a risk of cancer, or a cancer patient, but is not limited thereto.

As used herein, the term "leukapheresis" refers to selectively removing (separating) leukocytes from the collected blood and then transfusion to the patient again. In the case of leukocytes isolated by the above method, Ficoll- It can be used in the present invention without a separate method such as the Ficoll-Hypaque density gradient method.

As used herein, the term "peripheral blood mononuclear cell" may be used in the same sense as "PBMC", "mononuclear cell" or "monocyte", which is commonly used for anticancer immunotherapy It means a mononuclear cell separated from peripheral blood (Peripheral Blood). Peripheral blood mononuclear cells can be obtained by using a known method, such as a Ficoll-Hypaque density gradient separation method (Ficoll-Hypaque density gradient method) from the blood of a person who has taken blood.

The peripheral blood mononuclear cells are not necessarily autologous, and allogeneic peripheral blood mononuclear cells may be used for the production of high-purity natural killer cells for anticancer immunotherapy according to the present invention.

In addition, the peripheral blood mononuclear cells are not necessarily derived from normal people, and peripheral blood mononuclear cells of cancer patients may also be used for the production of high-purity natural killer cells for the anticancer immunotherapy according to the present invention.

The second separation step is performed by a method of separation using CD56 microbeads and/or CD3 microbeads, or a method of separation using equipment such as CliniMACSs, Flow Cytometry Cell Sorter, etc. can be

The separation method using the CD56 microbeads and/or CD3 microbeads is, for example, performed by adding CD56 microbeads to PBMC and then removing non-specific binding, or by adding CD3 microbeads to PBMC. It can be carried out by adding CD56 microbeads to remove specific binding, and then adding CD56 microbeads to remove non-specific binding.

T cells and the like can be effectively removed through the separation of the CD56+ cells and the CD3-/CD56+ cells.

As used herein, the term “feeder cell” refers to a cell that cannot divide and proliferate, but has metabolic activity, and helps the proliferation of target cells by producing various metabolites.

Said feeder cells are irradiated Jurkat cells, irradiated EBV-LCL cells (Epstein-Barr virus transformed lymphocyte continuous line), and/or PBMC, HFWT, RPMI 1866, Daudi, MM-170, TCO-2, K562 or It may be a cell that has been genetically engineered to target K562 cells (eg, K562-mbIL-15-41BB ligand), but is not limited thereto.

As used herein, the term "Jurkat cell" or "Jurkat cell line" is a blood cancer (immortalized acute T cell leukemia) cell line, Dr. of the University of California at San Francisco. It is a cell line developed by Arthur Weiss. As a cell capable of producing IL-2 and expressing various chemokine receptors, it is a candidate for feeder cells for anticancer immunotherapy because it high-expresses MHC class I, a natural killer cell activity inhibitory factor, on the cell surface. It was a cell line that had no potential at all. Jurkat cells used in the present invention can be obtained from ATCC (ATCC TIB-152).

As used herein, the term "EBV-LCL cell" or "EBV-LCL cell line" refers to an Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL, Epstein-Barr virus transformed lymphocyte continuous line, DM Koelle et al., J Clin Invest, 1993: 91: 961-968), a B cell line created by in vitro infection of human B cells with Epstein-Barr Virus. The EBV-LCL cells used in the present invention can be directly prepared and used in a normal laboratory through a method of adding cyclosporin A during the process of infecting EBV in PBMCs.

The EBV-LCL cells can be prepared, for example, by the following method:

Put 30x10 ⁶ PBMC in 9mL of culture medium, put it in a T25 culture flask, and then add 9mL of EBV supernatant. After adding 80 µL of cyclosporin A at a concentration of 50 µg/mL, incubate at 37°C. After 7 days of culture, remove 1/2 of the supernatant, put a new culture medium, and add 40 μL of cyclosporin A. Repeat the same process as on the 7th day once every 7 days until the 28th day of culture. The cell line can be used after 28 days of culture, and from this point on, the culture medium is cultured without cyclosporin A.

The Jurkat cells and EBV-LCL cells can be used as feeder cells through irradiation.

The irradiated Jurkat cells and irradiated EBV-LCL cells were 1:0.1-5, 1:0.1-4, 1:0.1-3, 1:0.1-2, 1:0.1-1.5, 1:0.5-1.5. Alternatively, it may be included in a content ratio of 1:0.75-1.25, but is not limited thereto.

The irradiated Jurkat cells and irradiated EBV-LCL cells were treated with 50-500, 50-400, 50-300, 50-200, 50-150, 70-130, 80-120 or 90-110 Gy radiation, respectively. It can be obtained, but is not limited thereto.

As used herein, the term "cytokine (Cytokine)" includes both "first cytokine" and "second cytokine", and refers to immune-activating cytokines that can be used to induce peripheral blood mononuclear cells into natural killer cells. do.

The cytokine is IL (Interleukin)-2, IL-21, IL-15, Flt3-L (FMS-like tyrosine kinase 3 ligand), SCF (Stem Cell Factor), IL-7, IL-18, IL-4 , type I interferons, GM-CSF (Granulocyte-macrophage colony-stimulating factor) and / or IGF 1 (Insulin-like growth factor 1) may be, but is not limited thereto.

Specifically, the first cytokine is IL-2, IL-21, IL-15, Flt3-L, SCF, IL-7, IL-18, IL-4, type I interferons, GM-CSF and/or IGF 1 may be, and the second cytokine may be IL-21, but is not limited thereto.

The second cytokine may be added one or more times during culture 0-6 days.

Specifically, it may be carried out by adding the second cytokine once on the 0th and 3rd days of culture.

When co-cultured with feeder cells and the first cytokine, when the second cytokine is additionally added once or more for culture 0-6 days, superior proliferation and anticancer activity can be exhibited, and even after freezing and thawing processes NK cells can be produced in sufficient numbers for clinical application.

The first cytokine is 50-1,000, 50-900, 50-800, 50-700, 50-600, 50-550, 100-550, 150-550, 200-550, 250-550, 300-550, 350-550, 400-550 or 450-550 IU/mL, the second cytokine is 10-1,000, 10-500, 10-100, 20-100, 30-100, 40-100, 50-100 or 10- It may be used at a concentration of 50 ng/mL, but is not limited thereto.

On the other hand, the conventional natural killer cell proliferation method required high concentrations of various cytokines, but the natural killer cell proliferation method according to the present invention uses two types of feeder cells for high-purity CD56+ cells (including CD3-/CD56+ cells). Therefore, it is possible to proliferate high-yield and high-purity natural killer cells even if the cytokine is used at a low concentration.

The co-culture was performed with peripheral blood mononuclear cells and feeder cells (eg, Jurkat cells and EBV-LCL cells) 1:1-100, 1:1-90, 1:1-80, 1:1-70, 1 :10-65, 1:20-65, 1:30-65, 1:40-65, 1:50-65 or 1:55-65 may be included in a mixing ratio of, but is not limited thereto.

The co-culture may be performed in a medium, and the medium that can be used in this case may be any medium used for induction and proliferation of peripheral blood mononuclear cells into natural killer cells without limitation. As such a medium, for example, RPMI-1640, DMEM, x-vivo 10, x-vivo 20 or cellgro SCGM medium may be used. Other culture conditions, such as temperature, may follow the culture conditions of normal peripheral blood mononuclear cells.

The first culturing step is 0-45, 0-42, 0-40, 0-35, 0-30, 0-20, 0-19, 0-18, 0-17, 0-16, 0-15 or It may be performed for 0-14 days, but is not limited thereto.

step of freezing

The cells prepared by culturing in the first culture step may be recovered and then suspended in a medium containing FBS and DMSO, and then stored frozen. For example, it may be suspended in a medium containing 90% FBS and 10% DMSO and stored frozen, but is not limited thereto.

Specifically, the frozen storage may be to put the prepared cells in a container for freezing storage containing isopropyl alcohol, and then transfer and store the cells at a temperature of -192° C. or less after overnight in a cryogenic refrigerator. Preferably, the prepared cells may be stored under liquid nitrogen after the temperature is lowered using a controlled rate freezer (CRF), but is not limited thereto.

steps to defrost

In the freezing step, the frozen cells may be thawed. For example, cells stored frozen may be thawed using a 37° C. water bath, but the present invention is not limited thereto.

second incubation step

This step may be performed by treating the feeder cells one or more times.

Specifically, it may be carried out by treating feeder cells one or more times in a culture period of 14 days.

When feeder cells are added and cultured at least once every 14 days of culture, they can exhibit superior proliferative and anticancer activity, as well as sustain cell growth to produce sufficient number of NK cells for clinical application even after freezing and thawing. can be

This step may further include the step of adding a cytokine.

Specifically, this step may further include the step of adding the first cytokine.

In addition, this step may further include the step of adding the second cytokine one or more times during culture 0-6 days.

Content common between this step and the steps of the present invention described above will be omitted in order to avoid excessive complexity of the present specification.

According to one embodiment of the present invention, the method for mass production of natural killer cells of the present invention may be to repeat the following steps.

freezing;

thawing; and

A second culturing step of co-culturing by adding feeder cells.

In one embodiment of the present invention, when using the method of the present invention, it was confirmed that the cell culture is smoothly performed even after the freezing and thawing process, and it is expected that natural killer cells can be mass-produced by repeating the above steps.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a method for mass production of natural killer cells.

(b) the mass production method of the present invention can produce natural killer cells in a sufficient number for clinical application even through a cryopreservation process, so the prevention and treatment of cancer using natural killer cells, especially the allogeneic treatment effect It can be usefully used for the purpose of promoting

1a and 1b are results of confirming the proliferative capacity of NK cells prepared according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1. Preparation of CD56+ natural killer cells (NK cells)-1

First, PBMCs were isolated from blood using a Ficoll-Hypaque density gradient method, and then cells were counted.

1-1. CD56+ cell isolation

MACS buffer (1x PBS+0.5% HSA) was added to the counted PBMCs to suspend the cells, and CD56 microbeads (Miltenyi Biotec) were added so that 1-20 μL per 1.0x10 ^{7 PBMC cells were added, followed by 5-} Incubated at 2-8° C. for 30 minutes. After incubation, MACS buffer was added and mixed, and then the cells were precipitated by centrifugation (600xg). After centrifugation, the supernatant was removed, and MACS buffer was added to float the cells, and the cells were placed in a column coupled to a MACS separator. MACS buffer was passed through the column to remove non-specific binding. After separating the column from the MACS separator and transferring it to a 15 mL conical tube, MACS buffer was added to separate the CD56+ cells attached to the column.

1-2. CD3-/CD56+ cell isolation

Separately, MACS buffer (1x PBS+0.5% HSA) was added to the counted PBMCs to suspend the cells, and CD3 microbeads (Miltenyi Biotec) were added so that 1-20 μL per ^{PBMC 1.0x10 7 cells were added, followed by 5-30} Incubated at 2-8° C. for min. After incubation, MACS buffer was added and mixed, and then the cells were precipitated by centrifugation (600xg). After centrifugation, the supernatant was removed, and MACS buffer was added to suspend the cells, and the cells were placed in a column coupled to a MACS separator. The MACS buffer was passed through the column to recover CD3-cells.

MACS buffer (1x PBS+0.5% HSA) was added to the recovered CD3- cells to suspend the cells, and CD56 microbeads (Miltenyi Biotec) were added so that 1-20 μL per ^{CD3- cells 1.0x10 7 cells were added, followed by 5-} Incubated at 2-8° C. for 30 minutes. After incubation, MACS buffer was added and mixed, and then the cells were precipitated by centrifugation (600xg). After centrifugation, the supernatant was removed and MACS buffer was added to suspend the cells, and the cells were placed in a column coupled to a MACS separator. MACS buffer was passed through the column to remove non-specific binding. After separating the column from the MACS separator and transferring it to a 15mL conical tube, MACS buffer was added to separate the CD3-/CD56+ cells attached to the column.

1-3. primary culture

The CD56+ cells or CD3-/CD56+ cells isolated in Examples 1-1 and 1-2 were prepared in advance with 100 Gy irradiated feeder cells (Jurkat cells and EBV-LCL cells) together with IL-2 and IL-21, respectively. After putting into RPMI-1640 medium containing 10% of FBS added at a concentration of 500IU/mL and 50ng/mL, 37°C, 5% CO ₂ Co-cultured in an incubator.

On the 6th day of culture, inoculated into a 350mL bag based on the number of cells 1.0x10 ^{5 -2.0x10 6} /mL, incubated for 4 days, and on the 10th day of culture, the cell number was placed in a 1L bag based on 1.0x10 ^{5 -2.0x10 6 /mL} After inoculation, further culture was performed for 4 days. At this time, (CD56+ cells or CD3-/CD56+ cells):(Jurkat cells):(EBV-LCL cells) were cultured at a ratio of 1:30:30.

1-4. Secondary culture after freezing and thawing

On the 14th day of culture, the cultured cells were suspended in a solution containing 90% FBS and 10% DMSO, stored frozen at -192°C or lower, and thawed in a 37°C water bath according to the culture schedule.

Then, RPMI-1640 containing 10% FBS supplemented with 100 Gy irradiated feeder cells (Jurkat cells and EBV-LCL cells) with IL-2 and IL-21 at concentrations of 500 IU/mL and 50 ng/mL, respectively. After putting in the medium, 37 ° C. 5% CO ₂ Co-cultured in an incubator.

After thawing the culture on day 6 the cells can 1.0x10 ⁵ -2.0x10 ⁶ / mL was inoculated into a 350mL bag (bag) based on additional four days after the culture, the thawed cells can be cultured day 10 1.0x10 ⁵ -2.0x10 ⁶ / mL for the reference After inoculation into a 1L bag, it was further cultured for 4 days.

On the 14th day after thawing, IL-2 and IL-21 together with 100 Gy irradiated feeder cells (Jurkat cells and EBV-LCL cells) were added at a concentration of 500 IU/mL and 50 ng/mL, respectively, to sustain the growth of cells in culture. It was put in RPMI-1640 medium containing 10% of FBS added, and then co-cultured in a 37°C 5% CO2 incubator.

On the 20th day of culture after thawing, inoculate into a 1L bag based on the number of cells 1.0x10 ^{5 -2.0x10 6} /mL, and incubate for 4 days, and on the 24th day after thawing, the number of cells 1.0x10 ^{5 -2.0x10 6} /mL After inoculation into a 1L bag, it was further cultured for 4 days.

Finally, after thawing, on the 28th day of culture, the cells were inoculated into a 1L bag based on 1.0x10 ^{5 -2.0x10 6} /mL and further cultured for 3-6 days. At this time, (CD56+ cells or CD3-/CD56+ cells):(Jurkat cells):(EBV-LCL cells) were cultured at a ratio of 1:30:30.

Example 2. Preparation of CD56+ Natural Killer Cells-2

Natural killer cells were prepared in the same manner as in Example 1, except for the step of adding cytokines in Example 1-4.

comparative example. Preparation of natural killer cells excluding the cytokine treatment step

Natural killer cells were prepared in the same manner as in Example 1, except for the step of adding cytokine (IL-21) in Examples 1-3 and 1-4.

experimental example. Confirmation of proliferative capacity of NK cells

The proliferative capacity of NK cells cultured by the methods of Examples and Comparative Examples was confirmed.

As can be seen in Figure 1, when cytokines were not treated during the primary culture (Comparative Example), it was found that a sufficient number of NK cells for clinical application was not produced when the freezing and thawing processes were performed (Fig. 1a). . On the other hand, when using the method of the present invention, NK cells were prepared in sufficient number to be clinically applied even after the freezing and thawing process, and these results were obtained not only when the cytokine was treated after the freezing and thawing process (Example 1), but also In the case of no treatment (Example 2), the same was observed ( FIG. 1B ).

Claims (15)

A first culture step of co-culturing at least one selected from the group consisting of CD56+ cells and CD3-/CD56+ cells with feeder cells and a first cytokine;
freezing;
thawing; and
A method for mass production of natural killer cells, including a second culturing step of co-culturing by adding feeder cells,
The first culturing step is to add a second cytokine one or more times during culture 0-6 days, the method. The method of claim 1, wherein the first cytokine is IL-2, IL-21, IL-15, Flt3-L, SCF, IL-7, IL-18, IL-4, type I interferons, GM-CSF and At least one selected from the group consisting of IGF 1, the method. The method of claim 1 , wherein the second cytokine is IL-21. The method according to claim 3, wherein the IL-21 is added at a concentration of 10-50 ng/mL. The method of claim 1 , wherein the feeder cells include irradiated Jurkat cells and irradiated EBV-LCL cells (Epstein-Barr virus transformed lymphocyte continuous line). The method of claim 5, wherein the irradiated Jurkat cells and the irradiated EBV-LCL cells are included in a content ratio of 1:0.1-5. The method according to claim 5, wherein the irradiated Jurkat cells and irradiated EBV-LCL cells are each obtained by treatment with 50-500 Gy radiation. The method according to claim 1, wherein the co-culturing of the first culturing step is performed by mixing any one or more cells and feeder cells selected from the group consisting of CD56+ cells and CD3-/CD56+ cells at a mixing ratio of 1:1-100. . The method according to claim 1, wherein the first culturing step is performed for 0-17 days. The method of claim 1, wherein the feeder cells of the second culturing step are treated at least once. The method of claim 1, wherein the second culturing step further comprises adding a cytokine. 12. The method of claim 11, wherein the cytokine is IL-2, IL-21, IL-15, Flt3-L, SCF, IL-7, IL-18, IL-4, type I interferons, GM-CSF and IGF 1 At least one selected from the group consisting of, a method. The method according to claim 1, wherein the method repeats the steps of:
freezing;
thawing; and
A second culturing step of co-culturing by adding feeder cells. The method of claim 13, wherein the feeder cells of the second culturing step are treated at least once. The method of claim 13, wherein the second culturing step further comprises adding a cytokine.

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